Interpretive Summary: Brassica napus, canola, is an important oil seed crop grown extensively in North America and northern Europe with annual yields exceeding seven million metric tons. Canola is the world's third most important vegetable oil crop, in significant part due to the low levels of erucic acid and glucosinolates in canola oil. However, the level of chlorophyll (Chl) content is significantly higher than that found in other major vegetable oils and is the biggest quality impediment in the canola oil industry. The major objective of this study was to identify those steps in Chl degradation in maturing canola seeds that are disrupted by exposure to freezing temperatures early in seed development. The results show that freezing interferes with a key regulatory step, which is also the degradation event that makes chlorophyll lose its green color. By defining the mechanism of this freeze-induced interference, the work narrows the focus of future work toward solving the canola green seed problem.

Technical Abstract:
Under normal field growth conditions, canola (Brassica napus) seeds produce chloroplasts during early seed development and then catabolize the photosynthetic machinery during seed maturation, producing mature seeds at harvest that are essentially free of chlorophyll. However, frost exposure early in canola seed development disrupts the normal programming of chlorophyll degradation resulting in green seed at harvest thereby significantly devaluing the crop. Pheophorbide a oxygenase (PaO), a key control point in the overall regulation of chlorophyll degradation, was affected by freezing. Pheophorbide a, the substrate of PaO, accumulated during late stages of maturation in seeds exposed to freezing during early seed development. Freezing interfered with the induction of PaO activity that normally occurs in the later phases of canola seed development when chlorophyll should be cleared from the seed. Moreover, we found that the induction of PaO activity in canola seed was largely post-translationally controlled and it was at this level that freezing interfered with PaO activation. The increased accumulation of PaO transcript and protein levels during seed development was not altered by the freezing episode and the increase in PaO protein was small compared to the increase in PaO activity. We found that PaO could be phosphorylated and that phosphorylation decreased with increasing activity implicating PaO dephosphorylation as an important post-translational control mechanism for this enzyme. Two PaO genes, BnPaO1 and BnPaO2, were identified in senescing B. napus leaves but only BnPaO2 expression was detected in maturing seeds.